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STUDY OF DENDRITIC CELL TARGETING BY ENGINEERED LENTIVECTORS
by
April M Tai
________________________________________________________________________
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(CHEMICAL ENGINEERING)
May 2012
Copyright 2012 April M Tai

Dendritic cell (DC) vaccines have great potential as an emerging form of immunotherapy, as DCs are potent antigen-presenting cells, capable of triggering T cell and B cell responses. Our lab has previously developed an engineered lentiviral vector (LV) that is pseudotyped with a mutated Sindbis virus glycoprotein (SVGmu), which is capable of targeting DCs through Dendritic Cell-specific ICAM3-grabbing Nonintegrin (DC-SIGN), a receptor that is predominantly expressed by DCs. ❧ We hypothesized that SVGmu interacts with DC-SIGN in a mannose-dependent manner, and that increasing the amount of high-mannose structures on the glycoprotein surface could result in higher targeting efficiencies of LVs towards DCs. It is known that 1-deoxymannojirimycin (DMJ) can inhibit α1,2-mannosidase I, which is an enzyme that removes high-mannose structures during the glycosylation process. Thus, we investigated the possibility of generating LVs with enhanced capability to modify DCs by supplying DMJ during vector production. Through western blot analysis and binding tests, we were able to infer that binding of SVGmu to DC-SIGN is directly related to amount of high-mannose structures on SVGmu. We also found that the titer for the LV produced with DMJ (FUGW/SVGmu + DMJ) on 293T.DCSIGN, a human cell line expressing the human DC-SIGN antibody, was over four times higher than that of vector produced without DMJ. In addition, transduction of a human DC cell line, MUTZ-3, yielded a higher transduction efficiency for the LV produced with DMJ. ❧ In our next study, we aimed to elucidate the internalization and trafficking mechanisms of this viral vector through confocal microscopy of GFP-Vpr-tagged virus, drug treatments, and dominant-negative mutants of GTPases, which are necessary for endosomal functions. Using these tests, we demonstrated that our engineered lentiviral vector enters the cell via receptor-mediated clathrin- and dynamin-dependent endocytosis, and that microtubule networks were also involved in a productive infection. Fusion was low-pH-dependent and occurred in the early endosomal stage of transport. Autophagy was also examined for its effect on transduction efficiency. We observed that enhanced autophage activity reduced viral infectivity, while suppressed autophagy boosted transduction efficiency. This study gives us insight on the internalization and trafficking mechanisms used by our engineered vector and gives us tools to improve the efficiency of this platform. ❧ In our last study, we examined the ability of lentiviruses enveloped with an alphaviral envelope glycoprotein derived from Ross River virus (RRV) to mediate transduction of DCs. We found that RRV was only able to specifically mediate transduction of cells through DC-SIGN when the viral vectors were produced under conditions limiting glycosylation to high-mannose glycans. This suggests that these RRV-pseudotyped LVs can be used for DC-targeting, but would require specific conditions during vector propagation for effective targeting infections.

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STUDY OF DENDRITIC CELL TARGETING BY ENGINEERED LENTIVECTORS
by
April M Tai
________________________________________________________________________
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(CHEMICAL ENGINEERING)
May 2012
Copyright 2012 April M Tai